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. Author manuscript; available in PMC: 2024 Dec 11.
Published in final edited form as: Nat Microbiol. 2020 Jul 20;5(10):1247–1261. doi: 10.1038/s41564-020-0753-6

Fig. 4. Effects of Vpu on SUMOylation and PML-NBs.

Fig. 4

a, PLA for BLM-SUMO. WTK1(HR/3’) cells were transfected with WITO Vpu-AU1 expression or control. Dot plot shows distribution of BLM-SUMO2/3 PLA foci numbers per cell, bars represent means ±SEM, n=3 biologically independent experiments (Control n=72, WITO n=53), ****p<0.0001. Representative microscopic images on the right. b, Immunoprecipitation of endogenous BLM. WTK1(HR/3’) cells transfected as in 4a. The right panel shows BLM SUMOylation values of 3 independent IPs in the presence of Vpu relative to the control IP; bars represent means ±SD, *p=0.0452. c, PLA for RAD52-SUMO2/3. WTK1(HR/3’) cells treated as in 4a. Dot plot shows distribution of RAD52-SUMO2/3 PLA foci numbers per cell, bars represent means ±SEM, n=3 biologically independent experiments (Control n=47, WITO n=55), ****p<0.0001. d, Involvement of PML in HIV-1 regulated homologous repair. WTK1(HR/3’) cells transfected with wt or vpu* pHIV-1-NL4-3-env*-IRES-mCherry constructs plus shRNAs targeting PML. Bars represent means ± SEM, n=2 biologically independent experiments in triplicates, right Western blot for PML knockdown efficiency; *p=0.0313. e, PML staining of WTK1(HR/3’) cells transfected with NL4-3 Vpu expression or control plasmid. Dot plot presents raw foci values and bars represent means ±SEM, n=3 biologically independent experiments (Control n=178, WITO n=196), ****p<0.0001. Representative microscopic images on the right. f, PLA to assess PML SUMOylation. Cells transfected as in panel 4a. Dot plot shows raw foci values and bars represent means ±SEM, n= 3 biologically independent experiments (Control n=67, WITO n=52), ****p<0.0001. Right, representative microscopic images. g, Immunodetection of SUMOylated PML. Western blotting of WTK1(HR/3’) cells transfected with wt or vpu* pHIV-1-NL4-3-env* constructs using a PML-specific antibody. Right: bars represent means ± SEM of 4 independent blots; *p=0.0344. h, Immunodetection of SUMOylated PML in CD4+ T cells. Cells transfected with wt or vpu* pHIV-1-CH058-env*-IRES-BFP were lysed, blotted and blots incubated with antibody specific for SUMOylated PML. Right: bars represent means +/- SEM of PML-SUMO levels from 3 donors, *p=0.0389. i, Pearson correlation analysis between BLM and PML-NB. WTK1(HR/3’) cells transfected as in 4g plus NL4-3 or WITO Vpu. Immunofluorescence performed 24h later. N=4 biologically independent experiments exp, ~100 nuclei analyzed each, bars represent means ± SEM, **p=0.0039, ***p=0.001, ***p=0.0002, ***p=0.0005. Two sided Wilcoxon matched-pairs test in a, c, d, e, f, i. Two sided unpaired t-test in b, g, h.